Osteogenic Potential Of Cervical Intervertebral Disc Fibroblasts In Vivo And Vitro: A Histochemical And Cell Culture Study

Osteogenic potential of cervical intervertebral disc flbroblasts invivo and vitro: a histochemical and cell culture studySummary of Background Data. Intervertebral disc degeneration such as anular protrusion and disc herniation is a common cause of cervical spondylosis myelopathy. Some studies confirm that proliferation of chondrocytes replace normal anular cells during disc degeneration.In other words, progressive ossification differentiation of flbroblasts in anular is the pathological basic of cervical spondylosis myelopathy.Objectives. This study was conducted to explore the osteogenic potential of cervical intervertebral disc fibroblasts in vivo and vitro. To investigate the regulatory factors of rhBMP-2 and TNF- a on osteogenic phenotype of fibroblasts and discuss the condition that facilitates osteogenesis of fibroblasts.Methods. (1) CHTF (cervical hollow threaded fixator) filled with different materials of cancellous bone and anulus fibrosus was applied to the fusion of the bone segments in experiment goats. X-ray films and CT were taken after operation to observe the stability and fusion of the segments. Pathological study of CHTF slice were applied to find the differentiation of annulus fibrosus.(2) To establish the annulus fibroblasts cell lines of experiment goats in vitro and the biologic specificity was found. The fibroblasts were grown in incubation in the media of rhBMP-2 and TNF-a for about three weeks , then,the marker for osteogenic features were investigated by biochemistry, histochemistry observations.(S) The fibroblasts were isolated , and purified from the annulus fibrosus of the cervical spondylosis myelopathy patients. In the same way, to explore the regulatory factors to the potentiality in inducing osteogenesis by fibroblasts.Results. (1). X-ray films and CT scans showed that the stability of the operated segment obtained by CHTF technique. The callus was found around the CHTF after 6 weeks of operation, especially in the position between the endplate ofthe vertebrae and metal.Pathological study of CHTF filled with cancellous bone showed new cartillage and bone trabecula after 6 weeks of operation. After 12 weeks of operation, new bone trabecula was obvious and nocrosis cancellous bone was found. Pathological study of CHTF filled with cancellous bone and anulus flbrosus showed nocrosis tissue and a little fibrous cartilage aound the fibous after 6 weeks of operation. At the point of 12 weeks, new bone trabecula and cartilage were found aound the old trabecula or fibous. The CHTF only filled with anulus fibrosus were found fibrous cartilage and new cartilage during the experiment. The CHTF filled with nothing only found a little fibrous cartilage afer 12 weeks of operation.(2).It was found that rhBMPi and TNF-α had no effect on the proliferation of fibroblasts from both the experiment goats and the cervical spondylosis myelopathy patients. Using rhBMP2 or TNF-a could stimulate fibroblasts to secrete alkaline phosphatase and I collagen. It was found that the combined use of rhBMP2 and TNF-a or the single use of rhBMP2 could make fibroblasts to secrete osteocalin and the morphological changes of the fibroblasts were very obvious.The fibroblasts orientated themselves in a radiating pattern around the calcium granules.The fibroblasts were interwoven one on top of another in the form of multilayer structure and the extracellular matrix increased in size in forming larger nodules. Histochemical study of the nodules with specific new bone labeller (Alizarin red S) revealed positive reaction, denoting that the nodules produced by the fibroblasts were bone tissues.Conclusions: The results point out clearly that rhBMP2 can induce the osteogenic potential of annulus fibroblasts in vitro. The osteogenic of annulus fibroblasts play an important role in the pathological changes of cervical spondylosis myelopathy.